Like many viruses, HIV hijacks the host cell's apparatus for normal protein ubiquitination and degradation, using it to eliminate antiviral proteins. Understanding how a virus recruits and targets the ubiquitination complex is critical fo developing therapeutics to prevent it. One HIV protein responsible for this hijacking is Virion infectivity factor (Vif). Vif is intrinsically disordered but gradually loses flexibility as it bids more host proteins, a process that may be crucial for function and represent a therapeutic opportunity. I hypothesize that the flexibility of the initial Vif complex results from sampling of alternate conformations rather than entropic side chain motions that are reduced upon Cul5 binding. I propose to test this hypothesis by characterizing the structure and flexibility of Vif in complex with human ubiquitination proteins EloB, EloC, and CBF-?, and how this changes as Cul5 binds. I will use a combination of molecular dynamics (MD) simulations and NMR experiments to look at the complex structure and dynamics, as well as small molecule docking and scoring methods to design a chemical probe that binds preferentially to certain states of the Vif complex. In this project I will probe the biophysical properties of this flexible protein complex and reveal the mechanism by which HIV commandeers host cell ubiquitination machinery. Specific Aim 1: Characterize the structure and flexibility of the four-protein complex of Vif, CBF-?, EloB, and EloC. Approach: MD of the Vif complex when Cul5 is removed and NMR experiments on the partially labeled Vif complex bound to APOBEC3F. Specific Aim 2: Add Cul5 to the complex to elucidate its allosteric effects upon binding. Approach: MD simulations of Cul5 binding to the Vif complex NMR experiments on the Vif complex bound to Cul5. Specific Aim 3: Design a chemical probe of Vif complex formation. Approach: Use state of the-art computational techniques to map small molecule binding pockets on the five protein crystal structure, dock small molecule fragments, and score these fragments to guide ongoing fragment based screening. This proposal will be the first computational and NMR study of the flexible Vif complex structure and dynamics. The knowledge gained will facilitate future development of HIV drugs targeting Vif. The training I receive will complement my current expertise with several new skills, including NMR of partially labeled protein complexes, relaxation dispersion NMR, and small molecule docking and scoring.